Non-alcoholic fatty liver disease (NAFLD) is estimated to occur in one third of the general population and is a major predisposing factor in the pathogenesis of hepatic insulin resistance and type 2 diabetes (T2D). NAFLD occurs when lipid supply to the liver exceeds rates of lipid oxidation and lipid export. A number of therapies have been employed to reduce ectopic-fat accumulation in liver and hepatic insulin resistance, however these approaches have been met with limited success in the long-term and new therapies are required. In order to meet this great unmet need, our lab has recently developed a Controlled Release Mitochondrial Protonophore (CRMP) that is functionally liver-targeted and causes increased mitochondrial fat oxidation by promoting a subtle sustained increase in hepatic mitochondrial uncoupling activity. Importantly, we have previously demonstrated that CRMP safely reverses hypertriglyceridemia, fatty liver, steatohepatitis and liver fibrosis in rodent models of NAFLD/NASH/liver fibrosis and T2D without inducing hyperthermia, weight loss or any associated hepatic/systemic toxicities. Taken together, these rodent studies provide important proof of concept for the further assessment of CRMP as a novel therapeutic strategy for the treatment of NAFLD/NASH and T2D in a highly relevant non-human primate model of NAFLD and the dysmetabolic syndrome. Therefore, in the present proposal, we aim to determine the safety and efficacy of chronic CRMP treatment on the reversal of hypertriglyceridemia, NAFLD, and liver and muscle insulin resistance in dysmetabolic, spontaneously obese Rhesus monkeys. In addition, we will perform a comprehensive set of hepatic metabolic flux measurements using state-of-the-art liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance spectroscopy methods to directly assess the impact of chronic CRMP treatment on rates of hepatic mitochondrial fatty acid oxidation, gluconeogenesis, pyruvate dehydrogenase flux, pyruvate kinase flux and hepatic ketogenesis in this obese dysmetabolic monkey model of NAFLD. We hypothesize that chronic CRMP treatment will reverse NAFLD and improve whole-body insulin responsiveness in obese dysmetabolic Rhesus monkeys in a safe and effective manner by promoting increased rates of hepatic mitochondrial fatty acid oxidation independent of changes in food intake/body weight. Collectively, the results of this research will be highly impactful in that they will provide important proof-of-concept and safety data in a highly relevant clinical model of obese dysmetabolic non-human primates to support the development of novel liver-targeted mitochondrial uncoupling agents for the treatment of NAFLD/NASH and T2D in humans.